Apparatus and method for centrifugally separating blood and then forming a fibrin monomer

ABSTRACT

in a process of separating a liquid sample having phase portions of different densities by centrifugal separation, a phase separator container is employed. The phase separator container comprises a housing having concentric inner and outer cylindrical walls defining a longitudinal axis and a top wall and further a piston body constituting a bottom wall of the housing. The piston body defines together with the outer cylindrical wall, the inner cylindrical wall and the top wall, an annular chamber for receiving the liquid sample. The piston body is displaceable within the annular chamber for draining a phase portion separated from the liquid sample through a drain conduit means communicating with the annular chamber. The phase separation chamber further comprises a reaction chamber to which the phase portions exposed from the annular chamber is processed. The apparatus further includes a liquid supply means for supplying the liquid sample to the annular chamber, a motor means for rotating the phase separation container round its longitudinal axis at a rotational speed causing a separation of the liquid sample into the phase portions, and an actuator means for displacing the piston body within the annular chamber.

This application is a divisional application of U.S. application Ser.No. 08/742,793, filed Oct. 31, 1996, now U.S. Pat. No. 5,792,344, whichis a divisional application of U.S. Ser. No. 08/421,599 filed Apr. 12,1995, issued as U.S. Pat. No. 5,603,845, which is acontinuation-in-part-of U.S. application Ser. No. 08/155,984, filed Nov.19, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to novel methods, devices and apparati for thecentrifugal separation of a liquid into its components of varyingspecific gravities, and is more particularly concerned with a bloodseparation device useful, for example, in the preparation of componentsfor a fibrin sealant.

BACKGROUND OF THE INVENTION

The separation of a liquid into its fractions, or components of varyingspecific gravity, has been carried out, inter alia, by centrifugation inmany hospital, laboratory and industrial settings. For example,centrifugation is widely used in blood separation techniques to separateblood into fractions containing plasma, platelets, red blood cells whiteblood cells and/or formed components, e.g. fibrinogen, fibronectin,factor VIII, factor XIII and the like. Quite simply, devices for use insuch techniques rely on the more dense components, e.g. thecell-containing fraction(s) in blood, being forced to a distal portionof the apparatus by the centrifugal force.

Many of the numerous device designs which utilize centrifugation can beplaced into two categories: a first group in which the sample containeris swung about a central axis of the centrifuge system itself; and, asecond group in which the chamber is rotated about its own longitudinalaxis. In the first category the container is typically a plastic bag ortube closed on one end. Such containers are orbited about the centralaxis of the centrifuge system such that the more dense components areforced to the bottom of the tube or to one side of the bag. Means arethereafter provided to selectively remove the less dense component, suchas plasma from the more dense component, such as blood cells andplatelets, or vice versa. Typically such means is a separator assemblywhich is insertable into an elongated blood-containing tube.Alternatively, when using a plastic bag, the bag is carefully squeezedso as to force out the plasma. U.S. Pat. No. 3,932,277 to McDermott etal discloses a device comprising a sample tube and a collection tube.The collection tube has a filter and check valve at one end which isinserted into an already centrifuged sample tube to collect the plasma.Similarly U.S. Pat. No. 3,799,342 to Greenspan utilizes a separatorhaving a check valve which opens upon pressurization of the samplecontainer to allow separated plasma to pass through into a collectionchamber. U.S. Pat. No. 4,818,386 to Burns employs a semi-buoyantseparator designed to have a specific gravity intermediate the specificgravities of two components into which the liquid is to be separated.Upon centrifugation, the separator moves within an elongated bloodsample tube to a position substantially between the more dense materialsat the bottom and the less dense materials at the top. An elastomericcup encompassing the separator locks the separator in place whencentrifugation is ceased to facilitate selective removal of the lessdense component.

As mentioned, a second category includes devices wherein theliquid-containing chamber is rotated about its longitudinal axis. Theliquid containing chamber is typically cylindrical or bowlshaped suchthat upon centrifugation heavier liquid components, e.g. blood cells,migrate outwardly toward the chamber wall and the lighter components,e.g. plasma, remain inward. Within this category are devices whichinclude conduits to other distinct containers, typically for the receiptand/or transfer of liquid during centrifugation, and devices which areself-contained for processing a fixed volume of liquid. One such deviceof the former variety is the "Latham bowl" disclosed and modified in anumber of patents including U.S. Pat. Nos. 4,086,924, 4,300,717, etc.The Latham bowl is designed such that the less dense components towardsthe inner portion of the spinning bowl are forced upward into acollection area inward of the outermost bowl radius. This system,however, requires a constant flow of blood to force the separated plasmaout and this "flow-during-spinning" feature mandates complex andexpensive rotary seals.

McEwen in U.S. Pat. No. 4,828,716 separates a liquid, such as blood,into its components, such as plasma and red blood cells, bycentrifugation in an elongated tube at speeds sufficient to provide aconcentric interface between these components. That is, a substantiallycylindrical apparatus is spun about its central or longitudinal axissuch that the more dense cellular components move to the outer wall andthe less dense components are inward of the more dense components.McEwen device thereafter reduces the volume of the processing chamberand collects the less dense plasma components by forcing it to a centralcollection port.

The above-described concentric separation occurs, by virtue of thecentrifugal, or G-force, acting upon the components, which is dependentupon radius and which can be expressed as

    G=1.18×10..sup.5 ×Radius (CM)×RPM.sup.2

To provide a good separation of components, it is beneficial to provideas "sharp" an interface as possible between the components of varyingdensity. Thus, for each liquid made up of two or more components, thereis minimal G-force needed to maintain this concentric interface. Onepotential difficulty with such prior artreducing-volume/concentric-interface devices is that it becomesdifficult to maintain the desired separation interface because as thevolume is reduced and the plasma is collected, the height of theprocessing chamber is also decreasing. This provides, obviously, thatthe constant volume of cellular (more dense) material is forced inwardto a decreasing radius. Indeed this must occur with the prior art deviceto force the plasma material centrally towards the collection port.However, it can be appreciated that when the radius of cellular materialdrops below the critical value needed to maintain a concentric interfaceat a given speed, the interface becomes much less clearly defined, ifnot nonexistent, and collection of unwanted cellular material results.For the McEwen-type blood separation, the volume of pure plasma is notas critical as for certain ocher applications. Also, the McEwen-typedevice operated at ullracentrifugation ranges.

In more current technologies, it has become critical to be able toseparate blood components with a more reliable purity of separationresulting in a higher hematocrit value, i.e. ratio of to red blood cellsto the total volume of the sample. It is also highly desirable to beable to provide separation in shorter periods of time and with minimalneed for detection devices. Further, ultracentrifugation can exertexcessive shear forces on blood components which have undesirableeffects, e.g. hemolysis. It would be useful in many applications toprovide the above liquid separation benefits, especially at centrifugespeeds below 20,000 RPM, preferably in the 3,000-15,000 RPM andoptimally in the 5,000-10,000 RPM range. Typically, centrifuge speedsabove about 10,000 RPM results in severe journalling and bearingproblems especially relating to the problem of providing adequatelubrication.

An object of the present invention is to provide more accurate andefficient separation of liquid, in particular blood, into its phaseportions of different densities through the employment of improvedseparation techniques. A particular advantage of the present inventionis that a quick, efficient separation of liquid components can beaccomplished without the disadvantages, i.e., expensive, complexequipment and damage to components such as blood components, of anultracentrifugation system.

A particular feature of the present invention relates to the fact thatin accordance with the novel separation and collection techniquesaccording to the present invention, a blood sample may be used toprovide a Fibrin extraction to be used in the preparation of a tissuerepair promoting substance, a so-called tissue-glue, which separationand preparation is carried out in a field compartment eliminating therisk that laboratory persons or operators are exposed to infectiousagents that may be passed through contact with blood, e.g. hepatitis oracquired immune deficiency syndrome.

A particular advantage of the present invention relates to the novelseparation and collection technique which renders it possible to performa separation of a blood sample for separating the blood sample intoplasma and blood cells which separation further provides a separation ofblood platelets from the blood cells and consequently provides theability to obtain plasma with a desired high or low platelet level byvarying the appropriate process parameters.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides for more accurate and efficientseparation of liquid into its separate components through the employmentof improved separation and collection techniques and novel devicessuitable in such techniques. Separation by centrifugation to provide aconcentric interface between components of a liquid is enhanced by usinga cylindrical housing having fixed outer and inner cylindrical wallwhich with top and bottom walls, define an annular chamber. The radiusof the inner cylindrical wall from the longitudinal axis of the deviceis chosen such that at the desired speed(s) of centrifugation there willalways be a sufficient centrifugal force (G-force) maintained at theinner cylindrical wall, and thereby throughout the annular chamber, tosustain such a concentric interface of components. By reducing thevolume of the annular chamber during centrifugation the desiredcomponent or compounds can be selectively removed via a drain means.

The resulting separation can be carried out relatively quickly and atrelatively low speeds. By a suitable choice of the inner and outerradius of the chamber, it is possible, for example, to achieve aseparation of up to 80% of the plasma of a blood sample in about 1minute at a speed of rotation of approximately 5,000 rpm. The axiallysymmetrical inner wall provides that the chamber in which the separationis to be carried out is annular, which in turn ensures that thecomponents in the chamber are always subjected to a G-force during thereduction of the volume which maintains a sharp interface. An annularchamber renders it furthermore possible in connection with a given bloodsample to achieve a relatively small distance between the inner wall andthe outer wall with the result that the components to be separated fromone another need only move a relatively short distance. Accordingly, theseparation is carried out quickly with a relatively high purity of theindividual components.

The above object, the above feature and the above advantage togetherwith numerous other objects, advantages and features which will beevident from the below description of presently preferred embodiments ofthe present invention are in accordance with the first aspect of thepresent invention obtained by an apparatus for separating a liquidsample having phase portions of different densities into said phaseportions by centrifugal separation, comprising:

a phase separation container, comprising:

a housing having concentric inner and outer cylindrical walls defining alongitudinal axis, a bottom wall, and a top wall, said outer cylindricalwall, said inner cylindrical wall, said bottom wall and said top walldefining together an annular chamber for receiving said liquid sample,

a piston body constituting said bottom wall or top wall of said housingand being displaceable within said annular chamber from a first positionin which a maximum interior volume is defined within said annularchamber to a second position in which a minimum interior volume isdefined within said annular chamber, and

a drain conduit means communicating with said annular chamber,

a liquid supply means for supplying said liquid sample to said annularchamber of said phase separation chamber as said piston body is in saidfirst position,

a motor means for rotating said phase separation container round saidlongitudinal axis at a rotational speed causing a separation of saidliquid sample into said phase portions,

an actuator means for displacing said piston body within said annularchamber from said first position towards said second position while saidphase separation container is rotated at said rotational speed so as toexpel one of said phase portions from said annular chamber through saiddrain conduit means.

The above object, the above feature and the above advantage togetherwith numerous other objects, advantages and features which will beevident from the below description of presently preferred embodiments ofthe present invention are in accordance with the second aspect of thepresent invention obtained by a phase separation container to be used inan apparatus for separating a liquid sample having phase portions ofdifferent densities into said phase portions by centrifugal separation,said phase separation container comprising:

a housing having concentric inner and outer cylindrical walls defining alongitudinal axis, a bottom wall, and a top wall, said outer cylindricalwall, said inner cylindrical wall, said bottom wall and said top walldefining together an annular chamber for receiving said liquid sample,

a piston body constituting said bottom wall or top wall of said housingand being displaceable within said annular chamber from a first positionin which a maximum interior volume is defined within said annularchamber to a second position in which a minimum interior volume isdefined within said annular chamber, and

a drain conduit means communicating with said annular chamber.

A third aspect of the present invention involves a cylindrical receiverchamber for receiving the phase portions expelled from the annularchamber and separated from the annular chamber by the piston body.

A fourth aspect of this invention pertains to the apparatus and phaseseparation container as above wherein the inner cylindrical wall of theannular chamber is a cylindrical wall component of the piston body.

A fifth aspect of the invention involves methods for using the aboveapparatus for separation of a liquid into phase portions of differentdensities.

A sixth aspect of the invention relates to such a method wherein theration (r_(i) :r_(o)) of the radius of the inner wall r_(i) to theradius of the outer wall r_(o) is between about 0.3:1 and about 0.8:1preferably 0.5:1.

A seventh aspect of the invention involves the apparatus and phaseseparation container as described above including connector meansconnecting the phase separation container to the motor means.

The above object, the above feature and the above advantage togetherwith numerous other objects, advantages and features which will beevident from the below description of presently preferred embodiments ofthe present invention are in accordance with another aspect of thepresent invention obtained by a method of separating a liquid samplehaving phase portions of different densities into said phase portions bycentrifugal separation, said method comprising:

providing a phase separation container, comprising:

a housing having concentric inner and outer cylindrical walls defining alongitudinal axis, a bottom wall, and a top wall, said outer cylindricalwall, said inner cylindrical wall, said bottom wall and said top walldefining together an annular chamber for receiving said liquid sample,

a piston body constituting said bottom wall or top wall of said housingand being displaceable within said annular chamber from a first positionin which a maximum interior volume is defined within said annularchamber to a second position in which a minimum interior volume isdefined within said annular chamber, and

a drain conduit means communicating with said annular chamber,

supplying said liquid sample to said annular chamber of said phaseseparation chamber as said piston body is in said first position,

rotating said phase separation container round said longitudinal axis ata rotational speed causing the generation of a gravitational fieldwithin said annular chamber so as to separate said liquid sample intosaid phase portions at any location within said annular chamber,

displacing said piston body within said annular chamber from said firstposition towards said second position while said phase separationcontainer is rotated at said rotational speed so as to expel one of saidphase portions from said annular chamber through said drain conduitmeans.

The above object, the above feature and the above advantage togetherwith numerous other objects, advantages and features which will beevident from the below description of presently preferred embodiments ofthe present invention are in accordance with another aspect of thepresent invention obtained by a method of separating a liquid samplehaving phase portions of different densities into said phase portions bycentrifugal separation, said method comprising:

providing a phase separation container, comprising:

a housing having concentric inner and outer cylindrical walls defining alongitudinal axis, a bottom wall, and a top wall, said outer cylindricalwall, said inner cylindrical wall, said bottom wall and said top walldefining together an annular chamber for receiving said liquid sample,

a piston body constituting said bottom wall or top wall of said housingand being displaceable within said annular chamber from a first positionin which a maximum interior volume is defined within said annularchamber to a second position in which a minimum interior volume isdefined within said annular chamber, and

a drain conduit means provided at or near said inner cylindrical walland communicating with said annular chamber,

supplying said liquid sample to said annular chamber of said phaseseparation chamber as said piston body is in said first position,

continuously rotating said phase separation container round saidlongitudinal axis at a rotational speed causing one of said phaseportions to be separated from said liquid sample, and

displacing said piston body within said annular chamber from said firstposition towards said second position while said phase separationcontainer is rotated at said rotational speed so as to continuouslyexpel said one of said phase portions from said annular chamber throughsaid drain conduit as said one of said phase portions is separated fromsaid liquid sample.

The above object, the above feature and the above advantage togetherwith numerous other objects, advantages and features which will beevident from the below description of presently preferred embodiments ofthe present invention are in accordance with another aspect of thepresent invention obtained by an apparatus for separating a liquidsample having phase portions of different densities into said phaseportions by centrifugal separation, further including:

means for detecting the characteristics of one or both of the componentswithin said phase separation container during the separation process.

The methods according to the above aspects of the present invention likethe apparatus and the container according to the other aspects of thepresent invention, are preferably used for separating plasma from ablood sample. Thus, the apparatus, the phase separation container andthe method according to the present invention are advantageously andpreferably used for separating a blood sample into various constituentssuch as blood cells and plasma optionally including platelets oralternatively constituting platelet-free plasma.

Most preferably the apparatus and methods are employed to prepare acomposition containing fibrin monomer or non-crosslinked fibrin,optimally for use in a fibrin sealant.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will now be further described with reference tothe drawings, in which

FIG. 1 is a schematic and sectional view of a first embodiment of asample container of a centrifugal separation and processing apparatusimplemented in accordance with the teachings of the present invention.

FIGS. 2-10 are schematic and sectional views similar to the view of FIG.1 illustrating specific steps of a separation and extracting processwhen employing the first embodiment shown in FIG. 1.

FIG. 11 is a schematic and sectional view similar to the view of FIG. 1of a second embodiment of a sample container of a centrifugal separationand processing apparatus implemented in accordance with the teachings ofthe present invention.

FIGS. 12-18 are schematic and sectional views similar to the views ofFIGS. 2-10 illustrating specific steps of a separation and extractionprocess when employing the second embodiment shown in FIG. 11.

FIG. 19 is a schematic and sectional view of a third embodiment or aprototype embodiment of a sample container of a centrifugal separationand processing apparatus implemented in accordance with the teachings ofthe present invention.

FIG. 20 is a perspective and exploded view of a component of the thirdembodiment shown in FIG. 19.

FIG. 21 is a schematic and partly broken-away view of a centrifugalseparation and processing apparatus in which the sample container isreceived for performing the separation and extraction process in aautomatized or semi-automatized manner.

FIGS. 22a-22c are schematic and sectional views of a mechanism forarresting and fixating the sample container relative to the centrifugeseparation and processing apparatus, illustrating three steps of thearresting and fixating process.

FIGS. 22d and 22e are schematic and sectional views of a lid componentof the sample container communicating with optical detectors implementedin accordance with two alternative optical detector principals.

FIG. 23 is a diagrammatic view illustrating the dependency between thegravitational force at the inner and outer walls of the phase separationchamber of the sample container and the rotational speed of the samplecontainer.

FIG. 24 is a diagrammatic view illustrating the yield percentage of aspecific blood sample when separating the blood sample by means of thesample container according to the present invention in dependency of thetime of performing the separation process further illustrating twospecific yield curves corresponding to the yield of plasma of high andlow blood platelets content, respectively.

FIG. 25 is a diagrammatic view illustrating the dependency between thevolume of a blood sample to be separated by means of the samplecontainer of a centrifuge separation and processing apparatus accordingto the present invention and the time of performing the separationprocess.

DETAILED DESCRIPTION OF THE DRAWINGS

In FIG. 1, a first embodiment of a sample container implemented inaccordance with the teachings of the present invention is showndesignated the reference numeral 10 in its entirety. The samplecontainer 10 constitutes a unitary structure to be used in a centrifugalseparation and processing apparatus to be described below. Although theinvention herein is described throughout in terms of blood separation,preferably for the preparation of components suitable for a fibrin glue,it should be appreciated that the devices, apparati and methods hereincan be employed with any liquid separation application. The presentinvention is particularly suited for the separation of a blood sampleinto blood cells and plasma and for preparing a Fibrin extract from theplasma, e.g. in accordance with the technique described in U.S. Pat. No.5,750,657.

In U.S. Pat. No. 5,750,657 methods and compositions for a completelynovel fibrin glue are disclosed. Generally, U.S. Pat. No. 5,750,657discloses a method of forming a fibrin sealant comprising contacting adesired site with a composition containing fibrin monomers andconverting this monomer to a fibrin polymer concurrently with thecontacting step, thereby forming the sealant at the desired site. Theterm fibrin is understood to include fibrin I, fibrin II and des BBfibrin. U.S. Pat. No. 5,750,657 further discloses a method of forming afibrin monomer composition comprising the steps of:

a) contacting a composition containing fibrinogen with a thrombin-likeenzyme to form a non-crosslinked fibrin polymer;

b) separation the non-crosslinked fibrin polymer from the fibrinogencomposition. and

c) solubilizing the non-crosslinked fibrin polymer to provide acomposition containing fibrin monomer.

The thrombin-like enzyme can be thrombin itself or can be another enzymewith similar activity, e.g., Ancrod, Acutin, venzyme, Asperase,Botropase, Crotalase, Flavoxobin, Gabonase, or Batroxobin, withBatroxobin being preferred.

In accordance with a preferred embodiment of the present invention, theearlier-disclosed fibrin monomer preparation can be carried out in arapid, efficient and safe manner in a unitary two-(or more) chamberdevice. The present device provides for such fibrin monomer preparationin less than 30 minutes and is especially useful in single donor orpreferably autologous fibrin sealant preparations. The single donor orautologous fibrin monomer composition can be co-administered with analkaline buffer or distilled water preferably including a source ofcalcium ions.

The sample container 10 comprises a housing 12 composed of a cylindricalwall component 14, a top wall component 16 and a bottom wall component18. The top wall component 16 is provided with a central through-goingaperture 20 in which a piston component 22 extends and seals relative tothe central through-going aperture 20 of the top wall componenttypically by means of an O-ring sealing 24.

The wall components 14, 16 and 18 are joined together after the pistoncomponent 22 is received within the central through-going aperture 20 ofthe top wall component 16, by any convenient means, e.g. by means ofmeshing threads or by gluing the wall components together. Thus, thecylindrical wall component 14 and the bottom wall component 18 mayconstitute an integral structure which is connected e.g. by glue or bymeans of meshing threads to the top wall component 16. Alternatively,the top wall component 16 and the cylindrical wall component mayconstitute an integral structure which is connected to a separate bottomwall component. Further alternatively, the wall components 14, 16 and 18may constitute three separate components which are joined together bymeans of meshing threads or in any other appropriate matter, e.g. bygluing or welding the wall components together.

As can be seen the inner cylindrical wall 26 and the outer cylindricalwall 14 define the annular chamber in which centrifugation takes place.The radii of these walls from the longitudinal axis of the container 10are chosen so that at the desired speeds of rotation a sufficientG-force is created to maintain concentric separation of the liquidcomponents. Of course, this will vary depending upon the liquid and thedesired speeds. For separating blood, for example, a G-force of about400 to about 1000 G should be maintained using the present apparatus.This provides that for speeds of about 5,000-10,000 RPM, preferablyabout 5,000 RPM, the radius of the inner cylindrical wall 26 istypically at least about 1.0 to about 1.5 cm depending upon the speedand blood sample. The radius of the outer cylindrical wall can varyaccordingly depending upon the sample size to be accommodated. Outerradii of about 2.0 to about 3.5 cm and above are suitable for separatingblood components in the 5,000-10,000 RPM range. Preferably the ratio ofinner wall radius r_(i) to outer wall radius r_(o) is from about 0.3:1to about 0.8:1 and most preferably about 0.5:1.

The piston component 22 comprises a cylindrical wall component 26 whichseals against the above mentioned O-ring sealing 24. The cylindricalwall component 26 is integrally connected to a circular plate component23, which is sealed relative to the inner surface of the cylindricalwall component 14 by means of an O-ring sealing 30. The O-ring sealings24 and 30 allow that the piston component 22 may be raised and loweredrelative to the housing 12 for varying the inner chambers defined withinthe sample container 10 as will be explained in greater detail below andfor sealing the inner chambers relative to one another and relative tothe environment.

The piston component 22 basically can define one, two or three chamberswithin the housing 12 of the sample container 10, vis a-vis firstchamber 32 which is of a basically annular configuration defined betweenthe cylindrical wall components 14 and 26, an optional second chamberdefined between the bottom wall component 18 and the circular placecomponent 28, and an optional third chamber 36 defined within thecylindrical wall component 26 of the piston body 22.

From the inner surface of the bottom wall component 18, an optionalprotrusion 38 constituted by one or more separate cam elements or acircular protrusion extends upwardly so that the inner volume of thesecond chamber 34 is not reduced to zero. Within the second chamber 34may be included a desired first chemical or biochemical agent 40 in anyform which can be used to treat or interact with a liquid componentseparated in the first chamber 32 and extracted into the second chamber34.

The piston component 22 can optionally be further provided with anannular lid component 42 which serves the purpose of receiving andsupporting an optional syringe 44. The syringe 44 can basically be aconventional disposable syringe comprising a cylindrical housing 46. Thesyringe 44 as described below in this preferred embodiment is useful forintroducing a desired second chemical or biochemical agent or solutioninto said second chamber 34. Alternatively, the syringe 44 may besubstituted by an ampulla or a syringe of a somewhat different structureand configuration for complying with specific requirements such asrequirements relating to mechanical compatibility relative to adispenser or syringe assembly in which the ampulla or the syringe is tobe used. At the uppermost end of the cylindrical housing 48, anoutwardly protruding annular flange 48 is provided and at the lowermostend of the cylindrical housing 46 a conical end tube 50 is providedwhich is connected to the cylindrical outer wall of the cylindricalhousing 46 through a bottom wall 52 of the cylindrical housing 46.Within the cylindrical housing 46, a plunger body 54 is received.

The conical end tube 50 can be received within a conical adaptor 56which communicates at its lower end with a tubing 58. The tubing 58 canbe of a substantial length as illustrated by the signature of the tubing58 allowing that the syringe 44 may be removed from the interior of thethird chamber 36 without disconnecting the conical end tube 50 of thesyringe 44 from the conical adaptor 56. The tubing 58 extends through acentral bore of the circular plate component 28 into the second chamber34 and communicates through a branch piece with a further tubing 60. Thetubing 60 communicates with an inlet 62 provided at the uppermost end ofthe cylindrical wall component 26 of the piston component 22 at whichoutlet a filter element 64 is provided. The tubing 60 constitutes aninlet tubing through which a second chemical or biochemical agent 88shown in FIG. 2 is introduced into an inner space defined within thecylindrical housing 46 of the syringe 44 which inner space is definedbelow the plunger body 54 as the plunger body is raised from a positionshown in FIG. 1 to the position shown in FIG. 2. After the introductionof the agent 88 into the inner space of the syringe 44, the inlet 62 ispreferably sealed by means of a sealing cap, not shown on the drawing.Alternatively, the inlet 62 may serve as a venting outlet for ventingany excess air of the tubing 58 and the tubing 60 into the atmosphere inthe process to be described below with reference to FIGS. 2-10.

The communication from the second chamber 34 to the tubing 58 mayinclude a microporous filter means 66 which is received within a recessprovided in the circular plate component 28 at the lower side surfacethereof. A desired biochemical or chemical agent can also be immobilizedor adsorbed onto said filter means 66 or elsewhere within said tubing 58to treat a first liquid component which is separated in the firstchamber 32 and extracted therefrom. From the first chamber 32communication is established to the second chamber 34 through a conduit65 which is established extending through the cylindrical wall component26 and the circular plate component 28 of the piston component 22. It isto be realized that the conduit 65 is shown provided at a radialposition within the outer surface of the cylindrical wall component 26.The conduit 65 can optionally be provided elsewhere in the upper surfaceof the piston plate 28 within the first chamber 32 according to whichliquid components are desired to be collected. The conduit 65 isnormally closed by means of a check valve 68 which may comprise asealing plug body 70 and a spring 72 journalled on a supporting seem 73and biasing the sealing plug body 70 toward a sealing or closingposition. Preferably, the check valve 68 is positioned as close aspossible relatively to the longitudinal axis of the sample container 10.Thus, in an alternative or modified embodiment of the sample container10, the check valve 68 is enclosed within a separate sub-chamberpositioned within the third chamber 36 and separated from the thirdchamber 36 through a separate wall component and further communicatingwith the first chamber 32 through a conduit extending through thecylindrical wall component 26 of the piston body 22. Alternatively, thecheck valve 68 may be housed within a separate recess provided withinthe circular plate component 28. The communication through the conduit65 into the second chamber 34 is established through a furthermicroporous filter element 74 similar to the microporous filter element66 described above. The microporous filter element 74 is received withinthe recess provided at the lower side surface of the circular platecomponent 28 of the piston component 22.

The first chamber 32 further communicates with a supply tubing 76through a bore 78 provided at the top wall component 16 of the housing12. The supply tubing 76 can be at its outer end provided with anadaptor for receiving a needle of a syringe (not shown in the drawings)containing a sample, preferably a blood sample to be introduced into thefirst chamber 32 of the sample container 10.

The first chamber 32 preferably communicates with the environmentthrough a venting tubing or conduit 82 establishing communication fromthe interior of the first chamber 32 to a venting outlet 84 providedopposite to the outlet 62 discussed above. The communication from thefirst chamber 32 through the venting tubing or conduit 82 is generallyestablished provided the piston component 22 is in the lowermostposition as shown in FIG. 1 as the inlet to the venting tubing orconduit 82 is raised above the O-ring sealing 24 provided the pistoncomponent 22 is raised to a position as shown in, e.g., FIG. 4.

Alternatively, vent means can be provided at any convenient locationwith said container 10.

The sample container 10 is as mentioned above preferably used forseparating a blood sample into blood cells and plasma having a highcontent of platelets or alternatively a low content of platelets andfurther for extracting a blood constituent from the plasma as will bedescribed below with reference to FIGS. 2-10.

In FIG. 2, a first step of a first process of separating a blood sample86 into specific liquid components and of separating a blood constituentfrom one of the liquid components is shown.

In FIG. 2, a blood sample 86 is contained within the first chamber 32and filling out a specific volume of the first chamber 32 providing aresidual air space 87 above the blood sample 86. In a preferredembodiment, the blood sample in the first chamber 32 is in the presenceof an anticoagulant. Any anticoagulant can be employed, and suitableexamples include heparin, EDTA, hirudin, cizrate and other calciumchelators such as NTA, HEEDTA, EDDHA, EGTA, DTPA, DCTA, HEPES, HIMOA,etc. The blood sample 86 contained within the first chamber 32 isdesignated by the plurality of small circles. Above the blood sample 86,an air space 87 is provided. In FIG. 2, the plunger body 54 of thesyringe 44 is raised and the redissolving buffer agent 88, which can be,e.g., a redissolving buffer solution and which has preferably beenintroduced into the interior of the syringe 44 as described abovethrough the tubing 60, is confined within the interior of the syringe44. The piston component 22 of the sample container 10 is in itslowermost position allowing that the first chamber 32 is vented throughthe venting tubing or conduit 82 as the blood sample 86 is introducedinto the first chamber 32. The redissolving buffer 88 is designated by aplurality of small triangles.

The redissolving buffer agent 88 can be any acid buffer solutionpreferably those having a pH between 1 and 5. Suitable examples, includeacetic acid, succinic acid, glucuronic acid, cysteic acid, crotonicacid, itaconic acid, glutonic acid, formic acid, aspartic acid, adipicacid and salts of any of these. Succinic acid, aspartic acid, adipicacid and salts of acetic acid, e.g., sodium acetate are preferred. Also,the solubilization may also be carried out at a neutral pH by means of achaotropic agent. Suitable agents include urea, sodium bromide,guanidine hydrochloride, KCNS, potassium iodide and potassium-bromide.Concentrations and volumes of such acid buffer or such chaotropic agentare as described in U.S. Pat. No. 5,750,657.

In FIG. 3, a second step of the first process is shown as the entiresample container 10 is rotated round the central longitudinal axis ofthe sample container 10. It is to be realized that the overall structureof the sample container 10 is of a basically symmetrical configurationas is evident from FIG. 1. It is further to be realized that the bloodsample contained within the first chamber 32 is exposed to a basicallyconstant centrifugal force of the order of 500-1,000 G as the firstchamber is of an overall annular configuration of a fairly small radialvariation and as the sample container 10 is rotated at a rotationalspeed of approximately 5,500 RPM. In FIG. 3, the blood sample containedwithin the first chamber 32 is separated into two components, a liquid90 containing blood cells and designated by the above described circles,and plasma 92 designated by a plurality of small squares. The liquid 90containing blood cells is of a somewhat higher density than the plasma92 causing a separation due to the high rotational speed generated asthe sample container 10 is rotating at a rotational speed of 5-10,000rpm.

As the sample container 10 is rotated at the above mentioned rotationalspeed, the check valve 68 is opened as the sealing plug body 70 isforced radially outwardly. Although the check valve 68 opens, the liquidcontained within the first chamber 32 does not flow through the conduit65, as on the one hand the liquid which is separated into the liquid 90and the liquid 92 is forced radially outwardly towards the cylindricalwall component 14 and as on the other hand the conduit 65 as pointed outabove, is provided at a radial position within the cylindrical wallcomponent 26. while the sample container 10 is still rotating, thepiston component 22 is in a third step of the first process raised fromthe position shown in FIG. 3 to the position shown in FIG. 4 causing atransfer of liquid from the first chamber 32 to the second chamber 34.

During the initial raising of the piston component 22, the air containedwithin the first chamber 32 is vented through the venting tubing orconduit 82. After the venting tubing or conduit 82 is raised above theO-ring sealing 24 any excess air of the air space 87 above in FIG. 2 ofthe first chamber 32 is transferred to the second chamber 34 as anycolumetric differences between the first chamber 32 and the secondchamber 34 are equalized through the venting tubing 60 communicatingwith the second chamber 34 through the microporous filter 66. The plasma92 shown in FIG. 3 is also transferred from the first chamber 32 to theoptional second chamber 34 through the conduit 65. In FIG. 4, the plasmatransferred to the second chamber 34 is designated the reference numeral94 and shown by squares as discussed above. As the volume of the secondchamber 34 is increased, the optional chemical or biochemical agent 40are also shifted from their positions shown in FIGS. 2 and 3 topositions at the inner cylindrical surface of the enlarged secondchamber 34. The agent 40, as discussed above, can be in any form, forexample, an agent or enzyme can be adsorbed or immobilized onto aparticulate substrate such as an enzyme bound to agarose gel or othersuch particles.

After a predetermined amount of plasma has been transferred from thefirst chamber 32 to the second chamber 34 or after substantial allplasma has been transferred from the first chamber Lo the second chamber34, the raising of the piston component 22 is stopped. It is to berealized that the microporous filter element 74 prevents that anyparticles such as blood cells may be transferred from the first chamber32 to the second chamber 34 in case the piston component 22 is raisedabove a position in which the plasma 92 has all been transferred fromthe first chamber 32 to the second chamber 34. It is further to berealized that the transfer of liquid from the first chamber 32 to thesecond chamber 34 and particular the state in which the plasma has beentransferred and the first chamber 32 contains blood cells exclusively iseasily detected by detecting the force which is employed for raising thepiston component 22 as the force required for transferring the bloodcells from the first chamber 32 to the second chamber 34 through themicroporous filter element 74 is far higher than the force required forraising the piston component 22 causing the transfer of the plasma fromthe first chamber 32 to the second chamber 34. The transfer of allplasma from the first chamber 32 to the second chamber 34 isconsequently easily detected as a radical increase in the force requiredfor further displacing the piston component 22.

Thereupon, in a fourth step of the first process the rotation of thesample container 10 is stopped as shown in FIG. 5. In FIG. 5, thesuspension of the agent 40 within the plasma 94 contained in the secondchamber 34 is allowed to react for a predetermined period of time. Forexample, an enzyme, e.g., Batroxobin, converts the fibrinogen from theplasma into fibrin monomer which almost instantaneously polymerizes intoa noncrosslinked fibrin polymer typically in the form of a gel as ismore clearly described in the aforementioned U.S. Pat. No. 5,750,657.

In a fifth and sixth step of the first process shown in FIGS. 6 and 7,respectively, the non-crosslinked fibrin polymer and the Batroxobinimmobilized in Agarose gel particles 40 and designated by a plurality ofsmall waves is separated from the plasma 94 contained within the secondchamber 34. The second chamber 34 may also contain an inner cylindricalwall so as to provide that the second chamber 34 is also annular. InFIG. 6, the sample container 10 is in the fifth step of the firstprocess rotated at a rotational speed causing a separation of a phase 96containing the non-crosslinked fibrin polymer gel and the bodies 40 fromthe plasma 94. The rotational speed at which the sample container 10 isrotated in the fifth step of the first process can be any speed but isconveniently in this process is somewhat lower than the afore-usedrotational speed at which the sample container 10 is rotated in thesecond step of the first process as described above with reference toFIG. 3, such as a rotational speed of approximately 0.5 times the aboverotational speed, i.e. a rotational speed of the order of 2,500RPM-3,000 RPM or lower. After the separation of the gel/partide phase 96from the plasma 94 contained within the second chamber 34, the pistoncomponent 22 is in the sixth step of the first process lowered causing atransfer of the plasma 94 from the second chamber 34 through the conduit65 to the first chamber 32 as the check valve 68 is opened. It is to berealized that the microporous filter element 74 prevents that anyparticles or larger bodies such as the agent particles 40 may betransferred from the second chamber 34 through the conduit 65 to thefirst chamber 32 as the microporous filter element 74 simply blocks thetransfer of particles or bodies.

After the completion of the second centrifugal separation and secondplasma transfer step, the second chamber 34 solely contains the liquid96 including the non-crosslinked fibrin polymer and the agent particles40 as shown in FIG. 7. Within the first chamber 32, a mixture 98 ofliquid containing the blood cell and the plasma retransferred from thesecond chamber 34 is contained as indicated by the combined symbols ofcircles and squares.

In FIG. 7 the redissolving buffer 88 is in a seventh step of the firstprocess added from the syringe 44 to the second chamber 34 by loweringthe plunger body 52 of the syringe 44 and simultaneously raising thepiston body 28 in order to provide a complete transfer of theredissolving buffer 88 from the syringe 44 to the second chamber 34 andpreventing that the redissolving buffer 88 is forced into the branchpiece of the tubing 58 and further into the venting tubing 60.

After a certain period of time during which the redissolving buffer 88causes the dissolving of the non-crosslinked fibrin polymer from theagent particles 40, forming a fibrin monomer-containing solution whichis to be transferred to the syringe 44 in an eighth and ninth step shownin FIGS. 9 and 10, respectively. The separation of the fibrin monomerfrom the Batroxobin in a liquid 100 produced in the second chamber 34through the action from the redissolving buffer 88 is simply carried outby any convenient separation process, e.g., through a filtering orpreferably a centrifugal separation process or a combination thereof asshown in FIG. 9. The centrifugal separation process is carried out inFIG. 9 through which the agent bodies 40 are separated from the liquid100 and collected at the inner side surface of the cylindrical wallcomponent 14 of the chamber 34 as the sample container 10 is rotated ata rotational speed which is normally smaller than the rotational speedsat which the sample container 10 is rotated in the separation stepsillustrated in FIGS. 3, 4 and 6 as the check valve 58 is not caused toopen for providing access through the conduit 65 from the second chamber64 to the first chamber 32. In FIG. 9, the liquid 98 contained withinthe first chamber 32 is clearly not exposed to a high gravitationalfield as the liquid is not on the one hand caused to be separated intoliquid components of different densities and on the other hand notshifted from the position also shown in FIG. 8 in which the liquidsurface is horizontal.

After the separation of the agent particles 40 from the liquid 100 in aninth step of the first process shown in FIG. 10 as discussed above withreference to FIG. 9, the liquid 100 is transferred from the secondchamber 34 of the sample container 10 to the syringe 44 contained withinthe third chamber 36 of the sample container by simultaneously raisingthe plunger body 54 of the syringe 44 and lowering the piston body 22.After the transfer of the fibrin monomer solution to the syringe 44 inthe ninth step of the first process shown in FIG. 10, the syringe 44 isremoved from the sample container 10 as a unitary structure integrallyconnected to the tubing 58 through the conical adaptor 56 on which theconical end tube 50 of the syringe 44 is received, as the tubing 58 isof a substantial length as discussed above. Thereupon, the syringe 44 isdisconnected from the sample container 10 as the tubing 58 is cut bymeans of a heating tool simultaneously causing a sealing of the free endof the tubing 58 connected to the conical adaptor 56. Consequently, theconical adaptor 56 serves the additional purpose of providing a sealingadaptor sealing the interior of the syringe 44 relative to theenvironment as the free end of the tubing connected to the conicaladaptor 56 is sealed. After the removal and disconnection of the syringe44 from the sample container 10, the remaining part of the samplecontainer 10 is disposed and destructed without spilling any liquidconstituents from the sample container which constituents might exposethe individual or individuals operating the centrifugal separation andprocessing apparatus on which the sample container 10 is processed tohazardous infection agents as bacteria or vira causing dangerousdiseases such as hepatitis or acquired immune deficiency syndrome.

As discussed above, this syringe 44 can be used to coadminister theso-produced fibrin polymer solution with appropriate alkaline buffer ordistilled water, preferably with a source of calcium ions, to provide afibrin sealant to a patient.

The above described sample container 10 and the above described firstprocess of separating a blood sample into specific liquid components andof separating a blood constituent from one of the liquid components maybe altered in numerous ways. First of all, the syringe 44 may be omittedas the third chamber 36 of the sample container 10 may constitute achamber in which the rediffusion buffer is initially contained orsupplied to in a step of the first process corresponding to the stepshown in FIG. 8 and into which the fibrin containing liquid 100 is lateron transferred in a final process step similar to the step shown in FIG.10.

The separation of the plasma in the step shown in FIG. 6 throughcentrifugal separation may be substituted by a simple filtering step inwhich the microporous filter element 74 is simply used for retaining theagent bodies 40 to which the fibrin is linked within the second chamber34 as the plasma is simply forced back into the first chamber 32.Similarly, the step of filtering the bodies 40 from the liquid 100 asshown in FIGS. 9 and 10 through centrifugal separation may besubstituted by a simple filtering separation step in which themicroporous filter element 66 is used for retaining the bodies 40 withinthe second chamber 34 as the redissolving buffer including the fibrin isforced into the syringe 44 or alternatively into the third chamber 36 ofthe sample container.

In FIG. 11, a second embodiment of a sample container implemented inaccordance with the teachings of the present invention is showndesignated the reference numeral 10' in its entirety. In FIG. 11 and theFIGS. 12-18 which illustrate specific steps of a second process ofseparating a blood sample into specific liquid components and ofseparating a blood constituent from one of the liquid components, whenemploying the sample container 10' in a second process very much similarto the process discussed above with reference to FIGS. 2-10 componentsor elements of the second embodiment of the sample container 10' whichcomponents or elements are identical to the components or elementsdescribed above with reference to FIGS. 1-10 are designated the samereference numerals as used in FIG. 1-10. The second embodiment 10' ofthe sample container basically differs from the above described firstembodiment in that the piston body 22 of the first embodiment 10 issubstituted by a piston body 22' of a slightly different configurationand structure. The piston body 22' comprises a cylindrical wailcomponent 26' and a circular plate component 28'. It can also be seenthat the cylindrical wall component 26' is recessed slightly at andabove the conduit 63' compared to the area of cylindrical wall 26' belowthe conduit 63'. The shoulder created herein helps keep blood cells outof the conduit 63' during the final stages of separation. Furthermore,the protrusion 38 shown in FIGS. 1-10 can be omitted as the chemical orbiochemical agent, e.g., Batroxobin immobilized in Aqarose gel, isprovided in a filtering container.

Within the piston body 22' of the second embodiment 10', the syringe 44is received within the third chamber 36 and communicates with the secondchamber 34 of the sample container through a tubing 58' similar to thetubing 58 shown in FIG. 1, however, differing from the tubing 58discussed above in that the branch piece establishing connection fromthe tubing 58 to the venting tubing 60 is omitted as the venting tubing60, the outlet 52 and the filter element of the outlet 52 are omitted.The communication between the first chamber 32 and the second chamber 34of the sample container 10' is also of a structure somewhat differentfrom the structure discussed above with reference to FIG. 1. Thecommunication including check valves which, however, are caused to openthrough the generation of a pressure difference and not a gravitationalforce as a clear distinction from the check valve 68 shown in FIG. 1 anddiscussed above.

The communication between the first chamber 32 and the second chamber 34of the sample container 10' includes two conduits. The first conduitincludes two conduit segments 63' and 65' and a first check valve 68interconnecting the conduit segments 63' and 65' and implemented as aball check valve including a ball 70' and allowing the transfer ofliquid from the second chamber 34 to the first chamber 32 and preventingthe transfer of liquid from the first chamber 32 to the second chamber34 through the first conduit. The second conduit includes two conduitsegments 63" and 65" and a check valve 68" implemented as a ball checkvalve including a ball 70" and further a container 69 in which an agent,e.g., Batroxobin is supported on a filter 66". It is to be realized thatthe inlet of the second conduit from the first container 32 is recessedrelative to the outlet of the first conduit 63' provided an annularchamber of somewhat reduced volume communicating with the second conduitexclusively further improving the accuracy of separating the plasma fromthe blood sample which is introduced into the sample container 10' aswill be described below with reference to FIGS. 12-18. The second checkvalve 68" allows the transfer of liquid from the first chamber 32 to thesecond chamber 34 and prevents retransfer of liquid from the secondchamber 34 to the third chamber 32 through the container 69. Thecommunication to and from the first chamber 32 through the first conduitcomprising the conduit segments 63' and 65' through the second conduitcomprising the conduit segments 63" and 65" is established through asingle microporous filter element 66 which is received within a centralrecess provided in the circular plate component 28' at the lowest sidesurface thereof.

The second embodiment 10' of the sample container is like the abovedescribed first embodiment 10 of the sample container preferably usedfor separating a blood sample into blood cells and plasma and furtherfor extracting a blood constituent from the plasma as will be describedbelow with reference to FIGS. 12-18.

In FIG. 12, a first step similar to the first step described above withreference to FIG. 2 of a second process of separating the blood sample86 into specific liquid components and of separating a blood constituentfrom one of the liquid components is shown.

In FIG. 13, a second step of the second process similar to the secondstep discussed above with reference to FIG. 3 is shown in which secondstep the plasma 92 is separated from the liquid 90 containing bloodcells.

In FIG. 14, a third step of the second process similar to the third stepdiscussed above with reference to FIG. 4 is shown in which third stepthe plasma 92 is transferred from the first chamber 32 to the secondchamber 34 through the second conduit comprising the conduit segments65" and 63" and further the check valve 68" and the container 69. As theplasma which is transferred from the first chamber 32 to the secondchamber 34 is contacted with the Batroxobin contained within thecontainer 69, the plasma contained within the second chamber 34 containsBatroxobin causing the conversion of fibrinogen from the plasma intofibrin monomer which immediately polymerizes into a non-crosslinkedfibrin polymer gel. The transfer of the plasma from the first chamber 32to the second chamber 34 has to be performed at a fairly low speed inorder to allow the plasma to react with the Batroxobin contained withinthe container 69. It should be understood that the speed with which thistransfer is made should correspond to the time necessary for thebatroxobin or other chemical agent to react with or treat the fibrinogenwithin the plasma.

After the transfer of the plasma 94 to the second chamber 34, andoptionally after a specific reaction period in which linking of fibringel is formed the sample container 10' may be rotated or may be stopped,the fibrin gel is separated from the remaining plasma liquid 94 andagent bodies 40 and in a fourth and fifth step of the second processshown in FIGS. 15 and 16, respectively, corresponding to the fifth andsixth step, respectively, of the second process discussed above withreference to FIGS. 6 and 7, respectively. The plasma 94 contained withinthe second chamber 34 of the sample container 10" is transferred fromthe second chamber 34 to the first chamber 32 through the first conduitcomprising the conduit segments 63' and 65' and the check valve 68'whereas the check valve 68" prevents that the plasma 94 is transferredthrough the second conduit.

In FIG. 17, a sixth step of the second process is shown in which stepthe redissolving buffer 88 is transferred to the fibrin containingliquid 100 by simply expelling the redissolving buffer 88 from thesyringe 44 in a manner similar to the step discussed above withreference to FIG. 8.

The second process of separating the blood sample into specific liquidcomponents and of separating a blood constituent, the fibrin of theblood sample from one of the liquid components when employing the samplecontainer 10' is finalized in a seventh step of the second process shownin FIG. 18 and corresponding to the ninth step shown in FIG. 10 bytransferring the fibrin-containing liquid 100 from the second chamber 34of the sample container 10' to the syringe 44 contained within the thirdchamber 36 of the sample container by simultaneously raising the plungerbody 54 of the syringe 44 and lowering the piston body 22'.Alternatively, the transfer of the liquid 100 from the second chamber 34of the sample container 10' to the syringe 44 may be controlled bydetecting the force which is used for moving the circular platecomponent 28' relatively to the housing 12 of the sample container 10'as described above with reference to the first embodiment of the samplecontainer implemented in accordance with the teachings of the presentinvention.

Like the first process described above with reference to FIGS. 2-10 andthe first embodiment of the sample container described above withreference to FIG. 1, the second process described above with referenceto FIGS. 12-18 and the second embodiment of the sample containerdescribed above with reference to FIG. 11 may be altered and modified innumerous ways, e.g. as discussed above. It is further to be realizedthat the first and second embodiments 10 and 10' described above withreference to FIGS. 1-10 and 11-18, respectively, may further be modifiedby simply turning the sample containers upside down in which case thesecond chamber 34 is positioned above the first chamber 32 and above thethird chamber 36.

In FIG. 19, a third embodiment of the sample container is shownconstituted by a prototype embodiment. The third embodiment of thesample container is designated the reference numeral 10" in itsentirety. The sample container 10" is of a structure basically identicalto the structure of the first and second embodiments 10 and 10'described above with reference to FIGS. 1 and 11, respectively. In FIG.19, components and elements which are identical to components andelements described above with reference to FIGS. 1 and 11 are designatedthe same reference numerals as used in FIGS. 1 or 11. The housing 12 ofthe third embodiment 10" differs from the housing 12 of the first andsecond embodiments 10 and 10', respectively, in that the cop wallcomponent 16" of the third embodiment comprises a skirt part 17" whichcircumferentially encloses the circumferential wall component 14establishing a sealed connection to the outer side surface of thecylindrical wall component 14. Through the top wall component 16", thebore 78 extends together with an additional conduit or bore 82" whichconstitutes a venting conduit similar to the conduit 82' described abovewith reference to FIG. 11 and communicating with a venting outlet 84"similar to the venting outlet 84' shown in FIG. 11. Alternatively, theventing outlet 84" may be closed by means of a closure or sealing cap,not shown on the drawings. Within the interior of the housing 12, apiston component 22" serving the same purposes as the piston components22 and 22' described above with reference to FIGS. 1 and 11,respectively, is received and sealed relative to the top wall component16 through the O-ring sealing 24 which seals against the peripheralouter wall of a cylindrical wall component 26" of the piston component22". The cylindrical wall component 26" constitute a length of a tubewhich is provided with outer threads at opposite ends for establishingconnection to a top flange component serving the purpose of supportingthe flange 48 of the syringe 44 which top flange component is not shownin the drawings and of meshing with interior threads of a cylindricalconnection piece which is integrally connected to a circular platecomponent 28" similar to the circular plate components 28 and 28'described above with reference to FIGS. 1 and 11. The junction betweenthe cylindrical connection piece 29 and the cylindrical wall component26 is sealed by means of an O-ring sealing 31.

Below the lower side surface of the circular plate component 28", amicroporous filter element 66" is arranged constituted by, for example,a piece of conventional cheese cloth, preferably supported on amicroporous filter constituting a composite filter structure. Themicroporous filter element 66" fulfills the same purpose as themicroporous filter element 66' shown in FIG. 11. Through the cylindricalwall component 26", two symmetrically arranged bores 27 extendestablishing communication from the chamber 32 circumferentiallyencircling the cylindrical wall component 26" to the interior of thepiston component 22".

At the lower end of the piston component 22", an assembly comprising aset of annular elements and a tubular element is supported within theinterior of the piston body 22". This annular assembly, shown herewithin the piston shaft but able to be located anywhere within this oranother centrifuge device, serves the purpose of filtering/chemicallytreating the liquid component separated in the first chamber 32.Generally, in annular concentric arrangement, this assembly comprises anoutermost annular support in body 108, and two annular filters spacedinwardly of the body 108 such that these open annular areas are defined

1) inward of the body 108;

2) inward of the first filter; and,

3) inward of the second filter.

More particularly, the assembly comprises a central component 102 whichis integrally connected to a tubular element 103 which is provided withouter threads at the upper end thereof which meshes with similarinternal threads of a fitting 56" which serves the same purpose as theconical adaptor 56 discussed above with reference to FIG. 1, vis-a-visthe purpose of receiving and establishing connection to the syringe 44.

The tubular component 103 is provided with a through-going bore 105 andfurther a transversal through-going bore 104 which is arranged inregistration with the through-going bore 27 of the cylindrical wallcomponent 26". The component 102 is fixated and sealed relative to thecircular place component 28" by means of two O-rings 106 and 107. Thecomponent 102 further serves the purpose of supporting an annularsupporting body 108 which is circumferentially sealed relative to theinner cylindrical surface of the cylindrical wall component 26" by meansof an O-ring 109. The supporting annular component 108 supports a set ofannular filtering elements 110 and 112 which together define an annularspace therebetween. The annular filtering elements 110 and 112 are, asis evident from FIG. 19, arranged in registration with the through-goingbores 27 and 104 of the cylindrical wall component 26" and the tubularelement 103, respectively. The annular filtering elements 110 and 112are further supported by an additional supporting component 114 which isprovided with a circumferential outer O-ring sealing 115 and which is ofa configuration similar to the configuration of the supporting annularcomponent 108. On top of the annular supporting component 114, a spacerelement 116 is provided, which spacer element is provided with internalthreads meshing with the outer threads of the tubular element 103.

The assembly described above with reference to FIG. 19 is shown inexploded view in FIG. 20.

The third embodiment of the sample container 10" shown in FIGS. 19 and20 is operated in a process similar to the processes described abovewith reference to FIGS. 2-10 and 12-13 for separating a blood sampleinto specific liquid components and for separating a blood constituentfrom one of the liquid components. The blood sample is as discussedabove introduced into the first chamber 32 and separated into a liquidcontaining blood cells and plasma through rotating the entire samplecontainer 10" round the longitudinal axis thereof at a high rotationalspeed providing centrifugal separation of the higher density blood cellsfrom the plasma. The plasma is transferred from the first chamber 32 tothe second chamber 34 by raising the piston component 22" while thesample container is rotated at the high rotational speed causing aninitial venting of excess air through the venting outlet 84" and atransfer of the plasma to the second chamber 34 through thethrough-going bores 27 and 104 of the cylindrical wall component 26" andthe tubular element 103, respectively, and the filtering elements 110and 112 positioned between the through-going bores 27 and 104 andfurther through the central through-going bore 105 of the tubularelement 103 to the microporous filter element 66". The Batroxobinimmobilized in the Agarose gels may be enclosed within the space definedbetween the annular filtering elements 110-112 which consequentlyconstitute a structure having a function similar to the container 69discussed above with reference to FIG. 11 or alternatively be enclosedwithin the second chamber 34 and supported on carrier Agarose bodiessimilar to the agent particles 40 described above with reference toFIGS. 2-10. After the extraction of Fibrin from the plasma, conversionof Fibrin into Fibrin 1, and linking of Fibrin 1 to Batroxobin, theplasma may be retransferred to the first chamber 32 in accordance withthe first process described above with reference to FIGS. 2-10 oralternatively be transferred to the syringe 44 through the activation ofthe plunger 54 causing the plasma to be forced into the interior of thesyringe 44.

In FIG. 21, an apparatus for receiving the sample container implementedin accordance with the teachings of the present invention and performingthe process of separating the blood sample into specific liquidcomponents and of separating a blood constituent from one of the liquidcomponents in an automatized or semi-automatized manner is disclosed anddesignated the reference numeral 120 in its entirety. The apparatuscomprises a housing 122 which is basically divided into threecompartments, an upper compartment 126, a central compartment 126 and alower compartment 128. The compartment 126 is preferablythermostatically controlled to a specific temperature and access to theinterior of the compartment 126 for positioning the sample container 10within the compartment and for removing the sample container and thesyringe 44 from the compartment 126 is obtained through an openableshutter or door 127.

Within the central compartment 126, the sample container 10 is receivedand supported on a rotatable turntable 130 which is journalled on ajournalling shaft 132 which constitutes an output shaft of a motor 134which is housed within the Lower compartment 123. The motor 134,consequently, constitutes a means for generating the high rotationalspeed at which the sample container 10 is rotated in specific steps ofthe above described process of separating a blood sample into specificliquid components and of separating a blood constituent from one of theliquid components.

In the upper compartment 144, two motors 136 and 138 are arrangedcooperating with vertically reciprocating actuator levers 140 and 142,respectively, cooperating with the plunger body 54 of the syringe 44 andthe annular lid component 42 of the piston body component 22,respectively.

The apparatus 120 further includes a control section 146 of the housing122 including an electronic circuitry, preferably a microprocessorcontrolled electronic circuitry which is operated by means of keys 148for initiating an controlling the operation of the apparatus 120 forperforming the above described process. The section 146 is furtherprovided with a display 150 on which the actual process step and anyrelevant information such as the duration of the process, thetemperature of the second compartment 126 of the housing 122 ispresented to an operator. The section 146 is preferably further providedwith interface means for interfacing the apparatus with an externalcomputer such as a personal computer and provided with detector meansfor detecting the overall operation of the apparatus including thetransfer of liquid from one of the above described chambers. Thedetection of liquid transfer may be based on optical detection ofconductivity detection involving the detection of constant or varyingelectric or magnetic fields. The detection of liquid transfer from thefirst chamber of the sample container to the second chamber of thesample container, from the second chamber of the sample container to thethird chamber of the sample container and from the second chamber of thesample container to the first chamber of the sample container mayalternatively be based on detection of the force transmitted to thepiston component as the force applied to the piston component increasesradically as the filter elements through which the liquid is to betransferred are blocked by blood cells or other larger bodies such asthe Agarose gel bodies. The above described embodiments of the samplecontainer constituting a separation component in which a blood sample isseparated into blood cells and plasma which is further processed for theprovision of a Fibrin extract constitutes a component in which a bloodsample provided from a patient is simple introduced into a first chamberof the sample container in which the entire separation and processingoperations are carried out without the necessity of human contact withthe blood sample or constituent thereof eliminating to any substantialextent the risk of exposing laboratory personnel or operators toinfectious agents from the blood sample which agents may cause diseasessuch as hepatitis or acquired immune deficiency syndrome. The Fibrinextract which is produced in accordance with the teachings of thepresent invention as described above is contained within a syringe whichis preferably used in a syringe dispenser of the type described ininternational patent application, application Nio. PCT/DK92/00287,international publication tio. WO93/06940, in which applicator thefibrin monomer containing liquid contained within the syringe 44 isneutralized through the mixture with a neutralizing agent. The processof separating plasma from a blood sample and of extracting or separatingFibrin from the plasma may e.g. be carried out in accordance with thetechniques described in the above mentioned EP 592,242.

The sample container 10 which is supported on the turntable 130 maypreferably be arrested and fixated relative to the turntable 130 bymeans of arresting or locking components which are shown in greaterdetails in FIGS. 22a, 22b and 22c. The locking components areconstituted by a downwardly protruding, circumferential rim partextension 160 of the cylindrical wall component 14 of the housing 12 ofthe sample container 10. The rim part extension 160 is provided withangularly spaced apart bores e.g. 900 or 1200 spaced apart bores one ofwhich is shown in FIGS. 22a-22c and designated the reference numeral162. The downwardly protruding, circumferential rim part extension 160is adapted to be received within a circumferential groove provided inthe top surface of the turntable 130. Within a radial bore extendingfrom the outer circumferential rim surface of the turntable 130, twolocking pins 166 and 170 are received. The pins 166 and 170 are biasedby means of springs 168 and 172, respectively, towards one another andare provided with blunt, conical end parts 167 and 171, respectively,which are contacted with one another at the center of thecircumferential groove provided in the top surface of the turntable 130unless the pins are moved apart as shown in FIG. 22a, as a lower endpart 164 of the circumferential, downwardly protruding rim partextension 160 is forced downwardly between the pins 166 and 170 causingthe pins to be separated from one another. The pins 166 and 170 and thesprings 163 and 172 are arrested within the radial bore of the turntableby means of a sealing plug 174 which is locked in position relative tothe circumferential outer rim surface of the turntable 130 by means ofmeshing threads, or any another appropriate locking structure.

In FIG. 22a, a first step of positioning the sample container 10relative to the turntable 130 is shown in which step the pins 166 and170, as described above, are forced apart as the lower end part 164forces the pins 166 and 170 apart allowing that the lower end part 164may pass downwardly relative to the pins 166 and 170.

In FIG. 22b, a second step of arresting the sample container 110relative to the turntable 130 is shown in which step the pins 166 and170 are forced into contact with one another within the throughgoingbore 162 of the downwardly protruding rim part extension 160 of thecylindrical wall component 14 of the housing 12. However, the samplecontainer may still be removed from the position shown in FIG. 2b bysimply raising the sample container causing the pins 166 and 170 to beseparated from one another as shown in FIG. 22a.

The mounting and removal of the sample container relative to theturntable 130 as shown in FIGS. 22a and 22b are accomplished while theturntable 130 is stationary. As the turntable 130 starts rotatingpropelled by the motor 134 of the apparatus shown in FIG. 21, the pins166 and 170 are acted upon by a centrifugal force which causes the pins166 and 170 to be shifted towards a radial offset position shown in FIG.22c, in which the pin 166 locks within the bore 162 of the rim partextension 160 preventing the housing 12 from being disconnected from theturntable 130 while the turntable and consequently the sample containerare rotated at the high and low rotational speeds during the processdescribed above with reference to FIGS. 1-18.

In FIGS. 22d and 22e, two alternative embodiments of optical detectormeans for detecting the transfer of liquid from the first chamber of thesample container to the second chamber of the sample container areshown. In FIGS. 22d and 22e, the top wall component 16 of the samplecontainer is of a conical configuration connected to an annular wallcomponent within which the cylindrical wall component 26 of the pistoncomponent 22 is received and sealed by means of the O-ring 24. Theannular wall component 17 is like the cylindrical wall component 26 ofthe piston component 22 preferably made from a light transparentmaterial allowing the transmission of light through the wall components.In FIG. 22d, the transfer of liquid from the first chamber of the samplecontainer is initiated and the plasma 92 is consequently forced into anarrow annular chamber defined between the outer surface of the wallcomponent 26 of the piston component 22 and the inner surface of thewall component 17. As the transfer of plasma from the first chamber ofthe sample container proceeds, the liquid 90 containing blood cells isforced into the above described narrow annular chamber as the plasma 92is transferred from the first chamber of the sample container. Thepresence of plasma or alternatively blood cells within the narrowannular chamber defined between the outer surface of the cylindricalwall component 26 of the piston component 22 and the inner surface ofthe annular wall component 17 is detected by means of optical detectormeans comprising a light generator 180 and an optical detector 133. Thelight generator 180 is positioned outside the annular wall component 17and includes a lamp 184 which is connected through an electric wire 182to the control section 146 of the apparatus 120 shown in FIG. 21. Thelamp 184 generates light which is focused by means of the focusing lens186 providing a substantially parallel light beam 192 which isirradiated to the above described annular chamber and the liquid presentwithin the annular chamber. Opposite to the light generator 180, anoptical detector 188 is positioned which is connected to the controlsection 146 of the apparatus 120 through an electric wire 190. Theoptical detector 188 receives the light transmitted from the lamp 184and focused by means of the focusing lens 186 through the abovedescribed annular chamber. The light generated by means of the lamp 184is optionally filtered for providing a substantially narrow lightspectrum which exhibit high transmission characteristics through plasmaand low transmission characteristics through blood cells in order toimprove the detection of blood cells within the annular chamber. Thelight generator 180 and the optical detector 188 may be supported withinthe second compartment 124 of the housing 122 described above withreference to FIG. 21 for irradiating light onto the above describedannular chamber and for detecting light received from the annularchamber, respectively.

In FIG. 22d, the presence of blood cells within the annular chamber isdetected in accordance with the light transmission detection technique.Alternatively, the presence of blood cells within the annular chambershown in FIGS. 22d and 22e may be detected in accordance with the lightreflection detection technique as shown in FIG. 22e.

In FIG. 22e, the light generator 180 and the optical detector 188 aresubstituted by an integral light generator and optical detector 180'including a lamp 184' similar Lo the lamp 184 shown in FIG. 22d and anoptical detector 188' similar to the optical detector 188 also shown inFIG. 22d. The lamp 184' and the optical detector 188' are connected tothe electronic circuitry of the apparatus 120 through electric wires182' and 190', respectively. The lamp 184' generates a light beam 192'which is irradiated to the annular chamber defined between the outersurface of the cylindrical wall component 26 of the piston component 22and the inner surface of the annular wall component 17. The wallcomponent 17 is like the wall component 17 described above withreference to FIG. 22d preferably made from a light transparent materialwhereas the cylindrical wall component 26 may be made from anon-transparent material, e.g., a light reflecting material. The lightirradiated to the liquid present within the annular chamber is partlyreflected as indicated by a light beam designated the reference number194'. The presence of blood cells within the annular chamber is inaccordance with the light reflection detecting technique detectedprovided the light which is irradiated to the annular chamber is partlyabsorbed by the red blood cells. Thus, the light generated by the lamp184' is preferably predominant green light which is reflected by theplasma 92 and absorbed by the red blood cells of the liquid 90. On thebasis of the shift of the detection signal generated by the opticaldetector 188', the presence of blood cells within the annular chamber isdetermined by the electronic circuitry of the control section of theapparatus 120.

EXAMPLE

A prototype embodiment of the sample container implemented as shown inFIGS. 19 and 20 was made from the following components:

The housing 12 of the sample container 10" was constituted by acylindrical housing component of an inner diameter of 70 mm, outerdiameter of 75 mm and a height of 80 mm. The bottom wall 18 of thehousing component 12 had a thickness of 2.5 mm. The housing component 12was cast from polymethylmethacrylate (PMMA). The lid component 17" wascast from POM and had an inner diameter of 75 mm, and outer diameter of80 mm and an axial height of 13 mm. The piston component 22" wasconstituted by a circular plate component 28" of an outer diameter of 70mm-0.1 mm, and a thickness of 7.4 mm. The sealing O-ring 30 was receivedwithin a groove of a height of 3.4 mm and a depth of 2.5 mm. Thecircular plate component 28" was also cast from PMMA. The cylindricalwall component 26" was made from a 100 mm length of a PMMA tube of aninner diameter of 30 mm. The wall component 26" was glued to thecircular plate component 28". The body 102, the tube 103, the body 108,the body 114 and the body 116 were all made from PMMA.

At a rotational speed of about 5,500 RPM the concentric separation ofplasma and red blood cells can be seen (viewed from top of container asdistinct concentric rings) almost immediately, i.e., within the firstminute. Over the next minute or two, the platelets can be seen leavingthe plasma as is evidenced by the lightening of the color of the plasma.For platelet-free plasma to be collected, the piston should not beraised until this complete separation has occurred. To collect plasmaincluding platelets, the piston should be raised immediately after redblood cell separation but before platelet migration. This is done by acontinuous raising of the piston during the platelet separation process.In this way the early portion of the sample collected is high inplatelets and the latter portion is low in platelets. Any desiredportion of such a sample or the entire platelet containing sample can beutilized as desired. Also, as would be apparent to those skilled in theart, plasma samples with specific platelet contents or specific puritiescan be collected by varying the speed, time of collection, amount ofcollection, etc.

In FIG. 23, a diagram is shown illustrating the dependency between thegravitational force generated within the first chamber 32 of theprototype embodiment 10" shown in FIGS. 19 and 20 and described in theabove example and the rotational speed at which the sample container isrotated. A curve A represents the gravitational force at the outer wallof the housing 12, i.e. adjacent to the inner side of the wall component14, and a curve B represents the gravitational force at the outer sideof the cylindrical wall component 26" of the piston component 22". It isevident from FIG. 23, that the gravitational force generated within theannular first chamber 32 is represented by. the area between the curvesA and B and further that the gravitational force at the outer wallcomponent 14 is approximately twice the gravitational force at thecylindrical wall component 26". Thus, a gravitational force varying lessthan approximately 2 is generated within the annular first chamber 32 asthe sample container is rotated.

In FIG. 24, a diagram is shown representing the dependency between thetime of rotating the prototype embodiment of the sample container shownin FIGS. 19 and 20 and described in the above example at a rotationalspeed of approximately 5,500 RPM and the percentage of a blood sample ofa volume of 90 ml which has been separated into plasma and blood cells.In FIG. 24, two curves C and D are shown representing the time ofseparation of the percentage of the blood sample for providingseparation of plasma from the blood cells which plasma includesplatelets as represented by the curve C and further separating theplatelets from the plasma as illustrated by the curve D. From FIG. 24,it is evident that an almost complete separation of the blood sampleinto blood cells and plasma has been accomplished after approximately1.5 minutes or even after approximately 1 minute as the blood cellsconstitutes approximately 15% of the blood sample which part cannot befurther separated. Provided the platelets are to be separated from theplasma, a complete separation of platelets free plasma is provided afterapproximately 3 minutes.

The separation of plasma including platelets from the blood sample ispreferably as mentioned above accomplished in a continuous process inwhich the piston body 22 of the first embodiment 10 or the piston body22' of the second embodiment 10' is raised continuously in order tocontinuously transfer the plasma from the first chamber 32 of the samplecontainer to the second chamber 34 of the sample container while thesample container is rotated at the high rotational speed causing theseparation of the blood sample into plasma and blood cells. Thecontinuous raising of the piston body is easily controlled by detectingthe transfer of plasma from the first chamber to the second chamberbased on the above described optical detector techniques oralternatively the detection of the force transmitted to the pistoncomponent for raising the piston body. Provided the transfer of plasmafrom the first chamber to the second chamber is carried out after acomplete separation of plasma from the blood sample has taken place, theplasma includes very few platelets and may even constitute platelet-freeplasma provided the centrifugal separation has been carried out for anextended period of time, i.e., about 3 minutes, as discussed above.

On the basis of the data represented in FIG. 24, a curve E isillustrated in a diagram shown in FIG. 25 illustrating the time requiredfor accomplishing a complete separation of a blood sample of a specificvolume in dependency of the time of rotating the above describedprototype embodiment of the sample container at the rotational speed of5,500 RPM. From FIG. 25, it is evident that within 60 seconds, a 90 mlblood sample may be separated into blood cells and plasma includingplatelets. A blood sample volume of the order of 100 ml constitutes amaximum blood sample which may be separated by means of the samplecontainer of the above example as the blood sample fills out a majorityof the annular first chamber of the sample container. Larger containers,as may be required, can easily be utilized within the scope of theteachings herein.

What is claimed is:
 1. An apparatus for separating blood into phaseportions having different densities by centrifugal separation andforming a fibrin monomer, comprising:a phase separation container havinga plurality of chambers, a drain conduit interconnecting two of saidchambers, liquid supply means for supplying said blood to a chamber,motor means for rotating said phase separation container at a rotationalspeed causing a separation of said blood into said phase portions, andan actuator means for forcibly expelling a phase portion of low densityfrom one of said chambers through said drain conduit into another ofsaid chambers; and processing means within said phase separationcontainer for processing said low density phase portion into a fibrinmonomer.
 2. A method of separating blood having phase portions ofdifferent densities into said phase portions by centrifugal separationand forming a fibrin monomer, said method comprising:providing a phaseseparation container having a plurality of chambers with a drain conduitinterconnecting two of said chambers; supplying said blood to one ofsaid chambers; rotating said phase separation container at a rotationalspeed causing a phase portion having a low density to be separated fromsaid blood; forcibly draining said low density phase portion from onechamber to another through said drain conduit; and processing said lowdensity phase portion within said chambers into a fibrin monomer.